Many analysis methods used in biology, chemistry, biotechnology, pharmaceutical and other research laboratories require accurate measurement and/or calibration of small volumes of liquids. These small volumes can range from nanoliters to milliliters. In one application, small volumes of liquid are dispensed from liquid delivery devices comprising a single delivery orifice or multiple orifices configured to deliver liquid simultaneously or sequentially. Specific examples include handheld multichannel pipettes, configured to deliver 8 or 12 channels at a time, and automated delivery equipment configured to deliver 96 or 384 channels at one time. In other applications, there is measured a precise amount of liquid contained by a small volume vessel.
For liquid delivery devices, the delivery must be both accurate and precise. At any given time the delivery device may not be functioning within the requirements of the process or the specifications of the manufacturer. For this reason it is necessarily to periodically calibrate the delivery equipment to ensure its correct operation and the integrity of the analysis.
Multi-channel delivery devices are typically used to expedite the analysis or processing of many samples at once, or to analyze one sample for many different attributes at once. In order to assure the integrity of the multi-analysis process, the equipment must be functioning correctly at the time of the analysis. Existing calibration methods have various limitations that prevent timely, convenient, accurate and/or precise calibration activities, thus bringing into question the results of the analysis. Several of these existing calibration methods are described below.
In the gravimetric method, a liquid volume is determined by weight. After initially weighing a receiving tube, the liquid volume is delivered into the tube. The tube is reweighed and the weight gain of the filled tube leads to calculation of the liquid volume, after correction for the density of the fluid, the loss of fluid due to evaporation during the procedure, and the buoyancy of air. The gravimetric method, however, is extremely time consuming, particularly for calibrating multiple liquid volumes as done with multi-channel liquid delivery devices where hundreds of liquid deliveries occur simultaneously. In the case of small delivery volumes, the errors introduced into the weighing by vibration, draft, static electricity, and/or evaporation cause this method to be of questionable utility or validity.
The gravimetric method can be applied to multiple volumes by the use of microtiter plates as the receiving vessel. Microtiter plates are rectangular molded plastic plates having a multiplicity of small cavities or wells to receive the liquid being delivered. Exemplary microtiter plates have 96 or 384 wells. In this method, the amount of liquid delivered to the wells is not measured individually. A complete microtiter plate is weighed without the liquid, the wells are filled and the plate is then re-weighed. The resulting weight gain is converted to liquid volume by accounting for liquid density, evaporation, and air buoyancy. The total volume contained by the microtiter plate is divided by the number of filled wells. Thus, this procedure measures the average liquid delivery volume. This method is disadvantageous for calibrating multiple-orifice delivery devices because no information is provided on the amount delivered from an individual orifice.
In the photometric method, a sample holder having a transparent bottom surface receives the liquid volume to be determined. A beam of light passes through the bottom surface and the liquid and eventually to a detector. The amount of light absorbed, i.e. the absorbance, provides information on the depth of the liquid and thus, the volume, taking into account certain properties of components of the liquid, such as concentration and molar absorptivity (or extinction coefficient). For example, if the liquid volume contains a dye capable of absorbing the light, the amount of liquid present can be estimated by measuring the amount of light absorbed as it passes through the well. The more liquid dispensed into a given well, the deeper the column of liquid, and the more light absorbed. Spectrophotometers for measuring the absorbance of the liquid by this technique are well known and are commonly called microplate readers or microtiter plate readers, where the photometric method is used with specially constructed microtiter plates. In the current implementation, however, the photometric method fails to provide an adequate level of accuracy for many uses.
Another means of calibration is to dispense liquid that contains a diluted amount of a fluorophore, such as fluorescein, into a microtiter plate. A specialized plate reader measures the amount of fluorescence coming from each well of the microtiter plate. As with the photometric method, this method is generally not sufficiently accurate to satisfy the needs of many users. There are no stable or recognized standards for calibrating the sensitivity of such fluorescent readers, making it impossible for this method to be used alone to provide a quantitative result traceable to a national standard (e.g., as set by a recognized standards organization, such as ASTM).